Journal: Experimental & Molecular Medicine

Article Title: Megakaryocytic TGFβ1 orchestrates osteogenesis of LepR + SSCs to alleviate radiation-induced bone loss

doi: 10.1038/s12276-025-01612-z

Figure Lengend Snippet: a Representative images of calcein and xylenol orange double labeling of bone and quantification of MAR and BFR for MK deleted mice and their littermate controls ( n = 6 mice per group). Scale bar, 100 µm. b Representative immunostaining images of LepR (red) in the BM of MK deleted mice and their littermate controls. The quantification of LepR + cells is shown in the right ( n = 6 mice per group). Scale bar, 100 µm. c Osteocalcin concentration in the BM of MK deleted mice and their littermate controls, determined by ELISA ( n = 6 mice per group). d Osteocalcin concentration in the serum of MK deleted mice and their littermate controls, determined by ELISA ( n = 6 mice per group). e PINP in the serum of MK deleted mice and their littermate controls, determined by ELISA ( n = 6 mice per group). f , g Representative immunostaining images of OCN (red) in the EB ( f ) and TB ( g ) of MK deleted mice and their littermate controls. The quantification of OCN + cells is shown on the right graphs ( n = 6 mice per group). Scale bar, 100 µm. h Quantitative biomechanical analysis of femora (peak load and stiffness) from MK deleted mice and their littermate controls ( n = 6 mice per group). i HE staining demonstrating metaphyseal bone and BM sections for MKs (black arrowheads at 48 h post-irradiation ( n = 6 mice per group). Scale bar, 100 µm. j Fluorescent images of mouse femoral bone. Lepr + cells (red) 7 days and 1 month after irradiation ( n = 6 mice per group). k Representative flow cytometry plots and quantification of percent MKs (CD41 + CD42d + ) and LepR + SSCs (Lepr + CD45 − CD31 − Ter119 − ) 7 days after irradiation ( n = 6 mice per group). Data on graphs are shown as mean ± SD. An unpaired two-tailed t -test was used to analyze the data in a – h and one-way ANOVA was used to analyze the data in j and k . * P < 0.05, ** P < 0.01 and *** P < 0.001. For all panels in this figure, data are representative of three independent experiments.

Article Snippet: Briefly, the bone sections were incubated with primary antibodies against mouse osteocalcin (A20800, ABclonal), Ki67 (AF7617, R&D), PTP1B (bs-55182R, Bioss), Slc39a14 (A10413, ABclonal), leptin receptor (bs-0410R, Bioss), CHOP ( A21902 , ABclonal), F4/80 (30325, CST), TGFβ1 (ab313729, abcam), vWF (bsm-52775R, Bioss), osterix (ab209484, abcam) and Smad2 (A7699, ABclonal) overnight at 4 °C and incubated with secondary antibodies for 1 h at 37 °C.

Techniques: Labeling, Immunostaining, Concentration Assay, Enzyme-linked Immunosorbent Assay, Staining, Irradiation, Flow Cytometry, Two Tailed Test

Journal: Experimental & Molecular Medicine

Article Title: Megakaryocytic TGFβ1 orchestrates osteogenesis of LepR + SSCs to alleviate radiation-induced bone loss

doi: 10.1038/s12276-025-01612-z

Figure Lengend Snippet: a Left: representative micro-CT images of longitudinal section femurs, cross-sectional view of the distal femurs and reconstructed trabecular structure of the region of interest from TGFβ1 MKΔ/Δ mice and their littermate controls (TGFβ1 fl/fl mice). Right: quantitative micro-CT analysis of the TB fraction (BV/TV, Tb.N, Tb.Th, Tb.Sp and Ct.Th) in TGFβ1 MKΔ/Δ mice and their littermate controls (TGFβ1 fl/fl mice) ( n = 6 mice per group). b LepR + SSCs were induced in osteogenic differentiation medium with or without MKs or (pretreated TGFβ type I receptor inhibitor SB431542) from wild-type (WT) mice after 14 days. Representative alkaline phosphatase staining images (left) and quantification of the activity of alkaline phosphatase was calculated (right) ( n = 6 per group). c LepR + SSCs were induced in osteogenic differentiation medium with or without MKs or (pretreated TGFβ type I receptor inhibitor SB431542) from WT mice after 21 days. Representative alizarin red staining images (left) and quantification of matrix mineralization was calculated (right) ( n = 6 per group). d LepR + SSCs were induced in adipogenic differentiation medium with or without MKs or (pretreated TGFβ type I receptor inhibitor SB431542) from WT mice after 21 days. Representative Oil O staining images (left) and the quantification of area was calculated (right) ( n = 6 per group). e qPCR analysis of the expression of Osterix , Runx2 , Adipoq and PPARγ in LepR + SSCs with or without MKs or (pretreated TGFβ type I receptor inhibitor SB431542) from WT mice after 7 days ( n = 3 per group). f LepR + SSCs were induced in osteogenic differentiation medium with or without MKs from the BM of TGFβ1 MKΔ/Δ and TGFβ1 fl/fl mice after 14 days. Representative alkaline phosphatase staining images and quantification of the activity of alkaline phosphatase was calculated ( n = 6 per group). g LepR + SSCs were induced in osteogenic differentiation medium with or without MKs from the BM of TGFβ1 MKΔ/Δ and TGFβ1 fl/fl mice after 21 days. Representative alizarin red staining images and quantification of matrix mineralization was calculated ( n = 6 per group). Data on graphs are shown as mean ± SD. One-way ANOVA was used to analyze the data in a – e and an unpaired two-tailed t -test was used to analyze the data in f and g . * P < 0.05, ** P < 0.01 and *** P < 0.001. For all panels in this figure, data are representative of three independent experiments.

Article Snippet: Briefly, the bone sections were incubated with primary antibodies against mouse osteocalcin (A20800, ABclonal), Ki67 (AF7617, R&D), PTP1B (bs-55182R, Bioss), Slc39a14 (A10413, ABclonal), leptin receptor (bs-0410R, Bioss), CHOP ( A21902 , ABclonal), F4/80 (30325, CST), TGFβ1 (ab313729, abcam), vWF (bsm-52775R, Bioss), osterix (ab209484, abcam) and Smad2 (A7699, ABclonal) overnight at 4 °C and incubated with secondary antibodies for 1 h at 37 °C.

Techniques: Micro-CT, Staining, Activity Assay, Expressing, Two Tailed Test

Journal: Experimental & Molecular Medicine

Article Title: Megakaryocytic TGFβ1 orchestrates osteogenesis of LepR + SSCs to alleviate radiation-induced bone loss

doi: 10.1038/s12276-025-01612-z

Figure Lengend Snippet: a RNA-seq analysis revealed changes in gene expression in LepR + SSCs co-cultured with MKs ( n = 3 each). b qPCR analysis of the expressions of S mad2 and Slc39a14 in LepR + SSCs, with or without MKs, from WT mice ( n = 6 per group). c Western blotting analysis of the expression of Smad2 and Slc39a14 in LepR + SSCs, with or without MKs, from WT mice ( n = 3 per group). d Representative immunostaining images of Smad2 (red) and Slc39a14 (green) in LepR + SSCs, with or without MKs, from the BM of TGFβ1 MKΔ/Δ and TGFβ1 fl/fl mice ( n = 6 per group). Scale bar, 100 µm. e Colocalization of Smad2 (red) with Slc39a14 (green) in LepR + SSCs, with or without MKs, from the BM of TGFβ1 MKΔ/Δ and TGFβ1 fl/fl mice ( n = 6 per group). Ctrl, control. f A schematic representation of the neural network model of Smad2 binding to the promoter region of Slc39a14, predicted by AlphaFold 3. g A plot of the predicted aligned error of the complex predicted by AlphaFold 3 (pTM + ipTM = 0.91). h A plot of the binding site and amino acid residues of Smad2–Slc39a14 analyzed by PyMol. i Dual-luciferase assays of 293T cotransfected with WT or mutated Slc39a14 (LUC), combined with pcDNA3.1-Smad2 or pcDNA3.1 vetor. j ChIP assay of Smad2 binding to Slc39a14 promoters in LepR + SSCs transfected with pcDNA3.1-Smad2 or pcDNA3.1. Immunoprecipitated DNA and the input DNA were detected by PCR. Primer sequences were designed for Slc39a14 promoter regions located in the promoter region of the Slc39a14 gene, with IgG as a negative control. Data on graphs are shown as mean ± SD. An unpaired two-tailed t -test was used to analyze the data in b and i . * P < 0.05, ** P < 0.01 and *** P < 0.001. For all panels in this figure, data are representative of three independent experiments.

Article Snippet: Briefly, the bone sections were incubated with primary antibodies against mouse osteocalcin (A20800, ABclonal), Ki67 (AF7617, R&D), PTP1B (bs-55182R, Bioss), Slc39a14 (A10413, ABclonal), leptin receptor (bs-0410R, Bioss), CHOP ( A21902 , ABclonal), F4/80 (30325, CST), TGFβ1 (ab313729, abcam), vWF (bsm-52775R, Bioss), osterix (ab209484, abcam) and Smad2 (A7699, ABclonal) overnight at 4 °C and incubated with secondary antibodies for 1 h at 37 °C.

Techniques: RNA Sequencing, Gene Expression, Cell Culture, Western Blot, Expressing, Immunostaining, Control, Binding Assay, Luciferase, Transfection, Immunoprecipitation, Negative Control, Two Tailed Test

Journal: Experimental & Molecular Medicine

Article Title: Megakaryocytic TGFβ1 orchestrates osteogenesis of LepR + SSCs to alleviate radiation-induced bone loss

doi: 10.1038/s12276-025-01612-z

Figure Lengend Snippet: a Serum zinc concentration in mice 4 weeks after irradiation with administration of TPO or vehicle ( n = 6 per group). b Representative fluozin-3 images and quantitative analysis of LepR + SSCs, with or without MKs, after irradiation ( n = 6 per group). c Representative immunostaining images of Slc39a14 (green) in LepR + SSCs, with or without MKs, after irradiation ( n = 6 per group). Scale bar, 100 µm. d KEGG enrichment analysis of upregulated pathways in LepR + SSCs after irradiation. e GO enrichment analysis of downregulated functions in LepR + SSCs after co-culture with MKs. The top ten enriched GO terms ( P < 0.05) are shown. f Western blotting analysis of the expression of Slc39a14, PTP1B, p-eIF2α, ATF4 and CHOP in LepR + SSCs after co-culture with MKs ( n = 3 per group). g Representative immunostaining images of CHOP (green) in LepR + SSCs, with or without MKs, after irradiation ( n = 6 per group). Scale bar, 100 µm. h Transmission electron microscopy images of LepR + SSCs after irradiation co-culture with MKs ( n = 3 per group). Data on graphs are shown as mean ± SD. One-way ANOVA was used to analyze the data in a – d and g . * P < 0.05, ** P < 0.01 and *** P < 0.001. For all panels in this figure, data are representative of three independent experiments.

Article Snippet: Briefly, the bone sections were incubated with primary antibodies against mouse osteocalcin (A20800, ABclonal), Ki67 (AF7617, R&D), PTP1B (bs-55182R, Bioss), Slc39a14 (A10413, ABclonal), leptin receptor (bs-0410R, Bioss), CHOP ( A21902 , ABclonal), F4/80 (30325, CST), TGFβ1 (ab313729, abcam), vWF (bsm-52775R, Bioss), osterix (ab209484, abcam) and Smad2 (A7699, ABclonal) overnight at 4 °C and incubated with secondary antibodies for 1 h at 37 °C.

Techniques: Concentration Assay, Irradiation, Immunostaining, Co-Culture Assay, Western Blot, Expressing, Transmission Assay, Electron Microscopy

Journal: Experimental & Molecular Medicine

Article Title: Megakaryocytic TGFβ1 orchestrates osteogenesis of LepR + SSCs to alleviate radiation-induced bone loss

doi: 10.1038/s12276-025-01612-z

Figure Lengend Snippet: a Representative immunostaining images of LepR (red) and PTP1B (green) in the BM of irradiated mice ( n = 6 mice per group). Scale bar, 100 µm. b Representative immunostaining images of LepR (red) and PTP1B (green) in the BM of MK deleted mice and their littermate controls after irradiation ( n = 6 mice per group). Scale bar, 100 µm. c Representative immunostaining images of PTP1B in LepR + cells, with or without, MKs from the BM of TGFβ1 MKΔ/Δ and TGFβ1 fl/fl mice after irradiation ( n = 6 per group). Scale bar, 100 µm. d Western blotting analysis of the expression of PTP1B and p-Stat3 in LepR + SSCs after co-culture with MKs ( n = 3 per group), inh = inhibitor. e Western blotting analysis of the expression of PTP1B and p-Stat3 in LepR + SSCs after co-culture with MKs from the BM of TGFβ1 MKΔ/Δ and TGFβ1 fl/fl mice ( n = 3 per group). Data on graphs are shown as mean ± SD. One-way ANOVA was used to analyze the data in c and d . * P < 0.05, ** P < 0.01 and *** P < 0.001. For all panels in this figure, data are representative of three independent experiments.

Article Snippet: Briefly, the bone sections were incubated with primary antibodies against mouse osteocalcin (A20800, ABclonal), Ki67 (AF7617, R&D), PTP1B (bs-55182R, Bioss), Slc39a14 (A10413, ABclonal), leptin receptor (bs-0410R, Bioss), CHOP ( A21902 , ABclonal), F4/80 (30325, CST), TGFβ1 (ab313729, abcam), vWF (bsm-52775R, Bioss), osterix (ab209484, abcam) and Smad2 (A7699, ABclonal) overnight at 4 °C and incubated with secondary antibodies for 1 h at 37 °C.

Techniques: Immunostaining, Irradiation, Western Blot, Expressing, Co-Culture Assay

Journal: Experimental & Molecular Medicine

Article Title: Megakaryocytic TGFβ1 orchestrates osteogenesis of LepR + SSCs to alleviate radiation-induced bone loss

doi: 10.1038/s12276-025-01612-z

Figure Lengend Snippet: a Representative micro-CT images of longitudinal section femurs, cross-sectional view of the distal femurs and reconstructed trabecular structure of the region of interest from mice injected with TPO or vehicle after irradiation ( n = 6 mice per group). b Quantitative micro-CT analysis of the TB fraction (BV/TV, Tb.N, Tb.Th, Tb.Sp, BMD and Ct.Th) in mice injected with TPO or vehicle after irradiation ( n = 6 mice per group). c Representative immunostaining images of OCN (red) in the TB and EB of the mice injected with TPO or vehicle after irradiation. The quantification of OCN cells is shown on the right ( n = 6 mice per group). Scale bar, 100 µm. d HE staining demonstrating B.Ar/T.Ar (bone area/total area) and the presence of MKs in the osteogenic niche of control or irradiated mice 8 weeks after injection with TPO ( n = 6 mice per group). Scale bar, 100 µm. e Von Kossa staining showing mineralization of bone matrix in control or irradiated mice 8 weeks after injected with TPO ( n = 6 mice per group). Scale bar, 1 mm. f Representative immunostaining images of perilipin (red) and osteopontin (OPN, green) in the BM of irradiation mice ( n = 6 mice per group). Scale bar, 100 µm. g Colocalization of LepR (red) with Slc39a14 (green) in the BM of irradiation mice ( n = 6 mice per group). Scale bar, 100 µm. Data on graphs are shown as mean ± SD. One-way ANOVA was used to analyze the data in b – g . * P < 0.05, ** P < 0.01 and *** P < 0.001. For all panels in this figure, data are representative of three independent experiments.

Article Snippet: Briefly, the bone sections were incubated with primary antibodies against mouse osteocalcin (A20800, ABclonal), Ki67 (AF7617, R&D), PTP1B (bs-55182R, Bioss), Slc39a14 (A10413, ABclonal), leptin receptor (bs-0410R, Bioss), CHOP ( A21902 , ABclonal), F4/80 (30325, CST), TGFβ1 (ab313729, abcam), vWF (bsm-52775R, Bioss), osterix (ab209484, abcam) and Smad2 (A7699, ABclonal) overnight at 4 °C and incubated with secondary antibodies for 1 h at 37 °C.

Techniques: Micro-CT, Injection, Irradiation, Immunostaining, Staining, Control

Journal: Experimental & Molecular Medicine

Article Title: Megakaryocytic TGFβ1 orchestrates osteogenesis of LepR + SSCs to alleviate radiation-induced bone loss

doi: 10.1038/s12276-025-01612-z

Figure Lengend Snippet: a Representative micro-CT images of longitudinal section femurs, cross-sectional view of the distal femurs and reconstructed trabecular structure of the region of interest from Slc39a14 leprΔ/Δ mice and their littermate controls (Slc39a14 fl/fl mice) ( n = 6 mice per group). b Quantitative micro-CT analysis of the TB fraction (BV/TV, Tb.N, Tb.Th, Tb.Sp, BMD and Ct.Th) in Slc39a14 leprΔ/Δ mice and their littermate controls (Slc39a14 fl/fl mice) ( n = 6 mice per group). c Quantitative biomechanical analysis of femora (peak load and stiffness) from MK deleted mice and their littermate controls (Slc39a14 fl/fl mice) ( n = 6 mice per group). d Serum zinc concentration in Slc39a14 leprΔ/Δ mice and their littermate controls (Slc39a14 fl/fl mice) ( n = 6 per group). e Von Kossa staining showing mineralization of bone matrix in Slc39a14 leprΔ/Δ mice and their littermate controls (Slc39a14 fl/fl mice) ( n = 6 per group). f Representative micro-CT images of longitudinal section femurs, cross-sectional view of the distal femurs and reconstructed trabecular structure of the region of interest from Slc39a14 leprΔ/Δ mice injected with TPO or vehicle after irradiation ( n = 6 mice per group). g Representative immunostaining images of OCN (red) in the TB and EB of the Slc39a14 leprΔ/Δ mice injected with TPO or vehicle after irradiation. The quantification of OCN cells is shown on the right ( n = 6 mice per group). Scale bar, 100 µm. h Representative immunostaining images of LepR (red) and PTP1B (green) in the BM of the Slc39a14 leprΔ/Δ mice injected with TPO or vehicle after irradiation. Scale bar, 100 µm. i Colocalization of LepR (red) with Ki67 (green) in the BM of the Slc39a14 leprΔ/Δ mice injected with TPO or vehicle after irradiation ( n = 6 mice per group). Scale bar, 100 µm. j Representative immunostaining images of TUNEL (green) in the BM of the Slc39a14 leprΔ/Δ mice injected with TPO or vehicle after irradiation. The quantification of tunel positive cells is shown on the right ( n = 6 mice per group). Scale bar, 100 µm. Data on graphs are shown as mean ± SD. An unpaired two-tailed t -test was used to analyze the data in b – d , g , i and j . * P < 0.05, ** P < 0.01 and *** P < 0.001. For all panels in this figure, data are representative of three independent experiments.

Article Snippet: Briefly, the bone sections were incubated with primary antibodies against mouse osteocalcin (A20800, ABclonal), Ki67 (AF7617, R&D), PTP1B (bs-55182R, Bioss), Slc39a14 (A10413, ABclonal), leptin receptor (bs-0410R, Bioss), CHOP ( A21902 , ABclonal), F4/80 (30325, CST), TGFβ1 (ab313729, abcam), vWF (bsm-52775R, Bioss), osterix (ab209484, abcam) and Smad2 (A7699, ABclonal) overnight at 4 °C and incubated with secondary antibodies for 1 h at 37 °C.

Techniques: Micro-CT, Concentration Assay, Staining, Injection, Irradiation, Immunostaining, TUNEL Assay, Two Tailed Test